Cerebrospinal Fluid Of Parkinson's Disease Patients Research Articles

Quantification of Circulating Cell-Free DNA in Idiopathic Parkinson's Disease Patients.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders globally and leads to an excessive loss of dopaminergic neurons in the substantia nigra of the brain. Circulating cell-free DNA (ccf-DNA) are double-stranded DNA fragments of different sizes and origins that are released into the serum and cerebrospinal fluid (CSF) due to cell death (i.e., necrosis and apoptosis) or are actively released by viable cells via exocytosis and NETosis. Using droplet digital polymerase chain reaction (ddPCR), we comprehensively analyzed and distinguished circulating cell-free mitochondrial DNA (ccf mtDNA) and circulating cell-free nuclear DNA (ccfDNA) in the serum and CSF of PD and control patients. The quantitative analysis of serum ccf-DNA in PD patients demonstrated a significant increase in ccf mtDNA and ccfDNA compared to that in healthy control patients and a significantly higher copy of ccf mtDNA when compared to ccfDNA. Next, the serum ccf mtDNA levels significantly increased in male PD patients compared to those in healthy male controls. Furthermore, CSF ccf mtDNA in PD patients increased significantly compared to ccfDNA, and ccf mtDNA decreased in PD patients more than it did in healthy controls. These decreases were not statistically significant but were in agreement with previous data. Interestingly, ccf mtDNA increased in healthy control patients in both serum and CSF as compared to ccfDNA. The small sample size of serum and CSF were the main limitations of this study. To the best of our knowledge, this is the first comprehensive study on serum and CSF of PD patients using ddPCR to indicate the distribution of the copy number of ccf mtDNA as well as ccfDNA. If validated, we suggest that ccf mtDNA has greater potential than ccfDNA to lead the development of novel treatments for PD patients.

Exogenous Tetranectin Alleviates Pre-formed-fibrils-induced Synucleinopathies in SH-SY5Y Cells by Activating the Plasminogen Activation System.

Parkinson's disease (PD) is a common neurodegenerative disease. Previously we identified tetranectin (TN) as a differentially expressed protein in the cerebrospinal fluid of PD patients, and we were surprised to find that TN knockout mice developed PD features. However, the specific role of TN in PD has not been clarified. In this study, we aimed to determine the effect of exogenous TN on cellular PD models and elucidate the underlying mechanisms. We found that exogenous TN could alleviate pre-formed-fibrils (PFFs)-induced synucleinopathies in SH-SY5Y cells and reduce the cell-to-cell transmission of α-synuclein (SYN). We also found that TN can promote the degradation of SYN by plasmin, which may account for its effect on cellular PD models. Moreover, administration of SYN/PFFs decreased the expression of TN and increased the expression of plasminogen activator inhibitor-1 (PAI-1) in SH-SY5Y cells, thereby reducing plasmin activity. Our findings depict a possible SYN-TN-plasmin interaction in which elevated levels of extracellular SYN monomers and aggregates in PD diminish the production of TN and PAI-1. Such changes lead to a reduced plasmin activity, which in turn reduces the degradation of extracellular SYN, thus forming a vicious cycle.

Oxidized/deamidated-ceruloplasmin dysregulates choroid plexus epithelial cells functionality and barrier properties via RGD-recognizing integrin binding

Choroid plexus epithelial cells (CPEpiCs) determine the composition of cerebrospinal fluid (CSF) and constitute the blood-CSF barrier (BCSFB), functions that are altered in neurodegenerative diseases. In Parkinson's disease (PD) the pathological environment oxidizes and deamidates the ceruloplasmin, a CSF-resident ferroxidase, which undergoes a gain of RGD-recognizing integrin binding property, that may result in signal transduction. We investigated the effects that oxidized/deamidated ceruloplasmin (Cp-ox/de) may exert on CPEpiCs functions. Through RGD-recognizing integrins binding, Cp-ox/de mediates CPEpiCs adhesion and intracellular signaling, resulting in cell proliferation inhibition and alteration of the secretome profile in terms of proteins related to cell-extracellular matrix interaction. Oxidative conditions, comparable to those found in the CSF of PD patients, induced CPEpiCs barrier leakage, allowing Cp-ox/de to cross it, transducing integrins-mediated signal that further worsens BCSFB integrity. This mechanism might contribute to PD pathological processes altering CSF composition and aggravating the already compromised BCSFB function.

Tryptophan Metabolites Are Associated With Symptoms and Nigral Pathology in Parkinson's Disease.

BackgroundThe objective of this study was to determine whether neurotoxic kynurenine metabolites, induced by inflammation, in plasma and cerebrospinal fluid (CSF) are associated with symptom severity and nigral pathology in Parkinson's disease (PD).MethodsClinical and MRI data were obtained from 97 PD and 89 controls. We used ultra‐performance liquid chromatography to quantify kynurenine metabolites and high‐sensitivity multiplex assays to quantify inflammation in plasma and CSF. We evaluated group‐wise differences as well as associations between the biomarkers, motor and nonmotor symptoms, and nigral R2* (MRI metric reflecting iron content).ResultsPD subjects had >100% higher 3‐hydroxykynurenine and 14% lower 3‐hydroxyanthranilic acid in plasma. The 3‐HK in plasma was closely associated with both symptom severity and disease duration. PD subjects also had 23% lower kynurenic acid in the CSF. Higher CSF levels of the excitotoxin quinolinic acid were associated with more severe symptoms, whereas lower levels of the neuroprotective kynurenic acid were linked to olfactory deficits. An elevated quinolinic acid/picolinic acid ratio in the CSF correlated with higher R2* values in the substantia nigra in the entire cohort. Plasma C‐reactive protein and serum amyloid alpha were associated with signs of increased kynurenine pathway activity in the CSF of PD patients, but not in controls.ConclusionsIn PD, the kynurenine pathway metabolite levels are altered in both the periphery and the central nervous system, and these changes are associated with symptom severity. Replication studies are warranted in other cohorts, and these can also explore if kynurenine metabolites might be PD biomarkers and/or are involved in PD pathogenesis. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Elevated serum mitochondrial DNA in females and lack of altered platelet mitochondrial methylation in patients with Parkinson´s disease

Purpose: Mitochondrial dysfunction has long been considered in the pathogenesis of Parkinson's disease (PD). This is evident from the presence of mitochondrial DNA deletions in substantia nigra neurons and respiratory chain abnormalities in the skeletal muscle of PD patients. However, the contributing factors that potentially cause oxidative stress in PD are still elusive. To a certain extent, the identification of acquired changes in circulating mitochondrial DNA (mtDNA) content in blood samples may mirror the mitochondrial (dys-) function. Therefore, herein, we investigated the mtDNA concentrations in serum and cerebrospinal fluid (CSF) of PD patients.Materials and methods: We performed quantitative analysis (qPCR) at two mitochondrial regions (D-Loop; ATPase6) and evaluated the platelet mtDNA methylation levels (MT-TL1 ,MT-CO1, MT-CO2 and MT-CO3) by bisulfite-PCR pyrosequencing.Results: Our quantitative analysis at two mitochondrial regions (D-Loop; ATPase6) revealed an increase in mtDNA serum concentrations in PD females compared to healthy females. Of particular interest, these altered concentrations were restricted to females serum only. Thus, in males as well as CSF of PD patients no increase was detected. Additionally, mtDNA methylation in platelets isolated from the plasma of PD patients showed no altered methylation levels in the mitochondrial MT-TL1 and MT-CO1 regions. Besides, a complete lack of platelet mtDNA methylation was observed at MT-CO2 and MT-CO3 mitochondrial sites.Conclusions: Taken together, we found an increased mtDNA serum concentration exclusively in PD females. As of yet, it is unclear whether this might reflect specific changes or characteristics of female PD pathobiology. However, in context to the ongoing debate about mtDNA methylation, we could show that the mitochondrial epigenome does harbor detectable CpG methylation sites in platelets-derived DNA.

Proteomics in Human Parkinson's Disease: Present Scenario and Future Directions.

Parkinson's disease (PD) is an age-related, threatening neurodegenerative disorder with no reliable treatment till date. Identification of specific and reliable biomarker is a major challenge for disease diagnosis and designing effective therapeutic strategy against it. PD pathology at molecular level involves abnormal expression and function of several proteins, including alpha-synuclein. These proteins affect the normal functioning of neurons through various post-translational modifications and interaction with other cellular components. The role of protein anomalies during PD pathogenesis can be better understood by the application of proteomics approach. A number of proteomic studies conducted on brain tissue, blood, and cerebrospinal fluid of PD patients have identified a wide array of protein alterations underlying disease pathogenesis. However, these studies are limited by the types of brain regions or biofluids utilized in the research. For a complete understanding of PD mechanism and discovery of reliable protein biomarkers, it is essential to analyze the proteome of different PD-associated brain regions and easily accessible biofluids such as saliva and urine. The present review summarizes the major advances in the field of PD research in humans utilizing proteomic techniques. Moreover, potential samples for proteomic analysis and limitations associated with the analyses of different types of samples have also been discussed.

SNAP25 Gene Polymorphisms Protect Against Parkinson's Disease and Modulate Disease Severity in Patients.

Parkinson's disease (PD) is a α-synucleinopathy in which intracellular aggregates of α-synuclein (α-syn) result in neurodegeneration and in the impairment of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex-mediated release of neurotransmitters. SNAP25 is a SNARE complex component: its concentration is increased in the cerebrospinal fluid of PD patients and this is related to the severity of cognitive and motor symptoms. Five SNAP25 single-nucleotide polymorphisms (SNPs) that modulate gene expression and were described to play a role in neurologic conditions (rs363050, rs363039, rs363043, rs3746544, and rs1051312) were analyzed in a cohort of 412 sporadic Italian PD patients and 1103 healthy controls (HC) in order to identify possible correlation with the disease. The SNAP25 rs1051312 C allele and CC genotype confer protection against PD onset, in particular in males (p = 0.003, OR(95%CI) = 0.67(0.51-0.88)) (pc = 0.008, OR(95%CI) = 0.28(0.10-0.70)). Co-segregation analyses revealed that the rs1051312 effect was reinforced when present within the rs363043 C-rs3746544 T-rs1051312 C haplotype (p = 3.3 × 10-4, OR = 0.47, 95%CI = 0.31-0.72), once again in males. Finally, rs363039 influenced age at onset (p = 0.02) and MMSE (Mini-Mental State Examination) scores (p = 0.01). The SNAP25 SNPs analyzed herein modulate gene expression at different levels as they are involved in binding miRNA and transcription factors; this suggests a possible synergistic effect of SNAP25 SNPs in the pathogenesis of PD. A replication in a larger and independent sample will help to further explore this hypothesis.

Cytokine profiling in the prefrontal cortex of Parkinson's Disease and Multiple System Atrophy patients

Parkinson's Disease (PD) and Multiple System Atrophy (MSA) are neurodegenerative diseases characterized neuropathologically by alpha-synuclein accumulation in brain cells. This accumulation is hypothesized to contribute to constitutive neuroinflammation, and to participate in the neurodegeneration. Cytokines, which are the main inflammatory signalling molecules, have been identified in blood and cerebrospinal fluid of PD patients, but studies investigating the human brain levels are scarce. It is documented that neurotrophins, necessary for survival of brain cells and known to interact with cytokines, are altered in the basal ganglia of PD patients. In regards to MSA, no major study has investigated brain cytokine or neurotrophin protein expression.Here, we measured protein levels of 18 cytokines (IL-2, 4–8, 10, 12, 13, 17, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1α and 1β, TNF-α) and 5 neurotrophins (BDNF, GDNF, bFGF, PDGF-BB, VEGF) in the dorsomedial prefrontal cortex in brains of MSA and PD patients and control subjects. We found altered expression of IL-2, IL-13, and G-CSF, but no differences in neurotrophin levels. Further, in MSA patients we identified increased mRNA levels of GSK3β that is involved in neuroinflammatory pathways. Lastly, we identified increased expression of the neurodegenerative marker S100B, but not CRP, in PD and MSA patients, indicating local rather than systemic inflammation. Supporting this, in both diseases we observed increased MHC class II+ and CD45+ positive cells, and low numbers of infiltrating CD3+ cells. In conclusion, we identified neuroinflammatory responses in PD and MSA which seems more widespread in the brain than neurotrophic changes.

Expression of prion protein in the cerebrospinal fluid of patients with Parkinson's disease complicated with rapid eye movement sleep behavior disorder.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases and mainly manifests with decreasing numbers of dopaminergic neurons. Rapid eye movement (REM) sleep behavior disorder (RBD) has an incidence of 15-47% in all PD patients. Prion proteins (PrPs), which are expressed in both neurons and glial cells of the brain, are believed to be correlated with abnormal neurological functions, although their role in PD-related sleeping disorders remains unclear. We therefore investigated the expressional profiles of PrP in PD patients with RBD. Quantitative real-time polymerase chain reaction and western blotting were used to detect the mRNA and protein levels of PrP, respectively, in the cerebrospinal fluid (CSF) of PD patients with RBD, PD patients without sleeping disorder, and healthy people (N = 23 each). We investigated the correlation between the CSF PrP level and sleeping behavior in PD patients. Patients with PD complicated with RBD had significantly elevated CSF PrP expression levels (both mRNA and protein) compared with either PD patients without sleeping disorder or healthy individuals (P < 0.05 in both cases). There is elevated expression of PrP in the CSF of PD patients with RBD. This may benefit the diagnosis of PD-related RBD.

Cdk5-Dependent Activation of Neuronal Inflammasomes in Parkinson's Disease.

Inflammasomes, which mediate the activation of caspase-1 and maturation of IL-1β and IL-18, have been unambiguously verified to participate in many diseases, such as lung diseases, infectious diseases and Alzheimer's disease, but the relation between Parkinson's disease and inflammasomes is poorly understood. The expression, maturation, and secretion of inflammasomes in neurons were measured. The activation of inflammasomes in the substantia nigra of the brain was tested in acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and an α-synuclein transgenic mouse model. The levels of IL-1β and IL-18 in cerebrospinal fluid and serum samples of Parkinson's disease (PD) patients and control subjects were measured. The role of cyclin-dependent kinase 5 (Cdk5) in neuronal inflammasome activation was evaluated using the pharmacological Cdk5 inhibitor roscovitine or Cdk5-targeted deletion. Here, we observed the expression of core molecules of inflammasomes, including NALP3, ASC, caspase-1, and IL-1β, in neuronal cells. The PD inducer rotenone could activate neuronal inflammasomes and promote the maturation and secretion of the cleaved IL-1β and IL-18 in a dose- and time-dependent manner. We also detected the activation of inflammasomes in the substantia nigra of a PD mouse model and in cerebrospinal fluid of PD patients. Furthermore, Cdk5 is required for the activation of inflammasomes, and both inhibition and deletion of Cdk5 could efficiently block inflammasome activation in PD models. Together, our results indicated that Cdk5-dependent activation of neuronal inflammasomes was involved in the progression of PD.

Parkinson's Disease with Fatigue: Clinical Characteristics and Potential Mechanisms Relevant to α-Synuclein Oligomer

Background and PurposeThe aim of this study was to identify the clinical characteristics and potential mechanisms relevant to pathological proteins in Parkinson's disease (PD) patients who experience fatigue.MethodsPD patients (n=102) were evaluated using a fatigue severity scale and scales for motor and nonmotor symptoms. The levels of three pathological proteins—α-synuclein oligomer, β-amyloid (Aβ)1-42, and tau—were measured in 102 cerebrospinal fluid (CSF) samples from these PD patients. Linear regression analyses were performed between fatigue score and the CSF levels of the above-listed pathological proteins in PD patients.ResultsThe frequency of fatigue in the PD patients was 62.75%. The fatigue group had worse motor symptoms and anxiety, depression, and autonomic dysfunction. The CSF level of α-synuclein oligomer was higher and that of Aβ1-42 was lower in the fatigue group than in the non-fatigue group. In multiple linear regression analyses, fatigue severity was significantly and positively correlated with the α-synuclein oligomer level in the CSF of PD patients, after adjusting for confounders.ConclusionsPD patients experience a high frequency of fatigue. PD patients with fatigue have worse motor and part nonmotor symptoms. Fatigue in PD patients is associated with an increased α-synuclein oligomer level in the CSF.

Ceruloplasmin functional changes in Parkinson's disease-cerebrospinal fluid.

BackgroundCeruloplasmin, a ferroxidase present in cerebrospinal fluid (CSF), plays a role in iron homeostasis protecting tissues from oxidative damage. Its reduced enzymatic activity was reported in Parkinson’s disease (PD) contributing to the pathological iron accumulation. We previously showed that ceruloplasmin is modified by oxidation in vivo, and, in addition, in vitro by deamidation of specific NGR-motifs that foster the gain of integrin-binding function. Here we investigated whether the loss of ceruloplasmin ferroxidase activity in the CSF of PD patients was accompanied by NGR-motifs deamidation and gain of function.ResultsWe have found that endogenous ceruloplasmin in the CSF of PD patients showed structural changes, deamidation of the 962NGR-motif which is usually hidden within the ceruloplasmin structure, and the gain of integrin-binding function. These effects occur owing to the presence of abnormal levels of hydrogen peroxide we detected in the CSF of PD patients. Interestingly, the pathological CSF's environment of PD patients promoted the same modifications in the exogenously added ceruloplasmin, which in turn resulted in loss of ferroxidase-activity and acquisition of integrin-binding properties.ConclusionsWe show that in pathological oxidative environment of PD-CSF the endogenous ceruloplasmin, in addition to loss-of-ferroxidase function, is modified as to gain integrin-binding function. These findings, beside the known role of ceruloplasmin in iron homeostasis, might have important pathogenic implications due to the potential triggering of signals mediated by the unusual integrin binding in cells of central nervous system. Furthermore, there are pharmacological implications because, based on data obtained in murine models, the administration of ceruloplasmin has been proposed as potential therapeutic treatment of PD, however, the observed CSF's pro-oxidant properties raise the possibility that in human the ceruloplasmin-based therapeutic approach might not be efficacious.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-015-0055-2) contains supplementary material, which is available to authorized users.

Tetranectin gene deletion induces Parkinson's disease by enhancing neuronal apoptosis

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). We previously identified tetranectin (TET) as a potential biomarker for PD whose expression is downregulated in the cerebrospinal fluid of PD patients. In the present study, we investigate the role of TET in neurodegeneration in vitro and in vivo. Our results showed that siRNA knockdown of TET decreased cell viability and the number of tyrosine hydroxylase (TH) positive cells, whereas it increased caspase-3 activity and the Bax/Bcl-2 ratio in cultured primary dopaminergic neurons. Overexpression of TET protected dopaminergic neurons against neuronal apoptosis in 1-methyl-4-phenylpyridinium cell culture model in vitro. In TET knockdown mouse model of PD, TET gene deletion decreased the number of TH positive cells in the SNpc, induced apoptosis via the p53/Bax pathway, and significantly impaired the motor behavior of transgenic mice. The findings suggest that TET plays a neuroprotective role via reducing neuron apoptosis and could be a valuable biomarker or potential therapeutic target for the treatment of patients with PD.

Serotonin impairment in CSF of PD patients, without an apparent clinical counterpart.

In Parkinson's disease (PD), several studies have detected an impaired serotonin (5-HT) pathway, likely affecting both motor and non-motor domains. However, the precise impact of 5-HT impairment is far from established. Here, we have used a HPLC chromatographic method, in a homogenous cohort (n = 35) of non fluctuating, non dyskinetic PD patients, to assess the concentration of 5-HT and its metabolite 5-HIAA in peripheral cerebrospinal fluid (CSF) obtained from lumbar puncture (LP). LP was performed following three days of therapy withdrawal, in order to vanish the effects of prolonged released dopamine agonists (DA), and in absence of any serotonergic agent. The PD patient group showed a significantly reduced CSF level of both 5-HT and 5-HIAA compared to either age-matched control subjects (n = 18), or Alzheimer's disease patients (n = 20). However, no correlation emerged between 5-HT/5-HIAA concentrations and UPDRS-III (r = −0.12), disease duration (r = −0.1), age (r = −0.27) and MMSE (r = 0.11). Intriguingly, low CSF 5-HT levels did not differ for gender or for motor phenotype (in terms of non-tremor dominant subtype and tremor dominant subtype). Further, low CSF 5-HT levels did not correlate with the presence of depression, apathy or sleep disturbance. Our findings support the contention that 5-HT impairment is a cardinal feature of stable PD, probably representing a hallmark of diffuse Lewy bodies deposition in the brainstem. However, clinical relevance remains uncertain. Given these findings, an add-on therapy with serotonergic agents seems questionable in PD patients, or should be individually tailored, unless severe depression is present.

Knockdown of interleukin-1 receptor 1 is not neuroprotective in the 6-hydroxydopamine striatal lesion rat model of Parkinson's disease

It is well established that neuroinflammation is associated with the progression of many neurodegenerative diseases, including Parkinson's disease (PD). Activated microglia and elevated levels of pro-inflammatory cytokines such as interleukin-1β (IL-1β) have been found in the brain and cerebrospinal fluid of PD patients, suggesting that IL-1β may be involved in the pathogenesis of this disease. This study aimed to knock down the expression of the interleukin-1 type 1 receptor (IL-1R1) to evaluate any potential therapeutic effect of limiting the action of IL-1β in the substantia nigra following a unilateral intrastriatal 6-hydroxydopamine (6-OHDA) lesion in rats. Adult Sprague-Dawley rats received intranigral injections of shRNA specific for IL-1R1, followed 2 weeks later by intrastriatal 6-OHDA. Injection of IL-1R1 shRNA did not prevent 6-OHDA-induced loss of motor function or loss of nigral dopamine neurons. IL-1R1 expression was increased in the midbrain following 6-OHDA injection; this effect was attenuated in 6-OHDA-treated animals that had received IL-1R1 shRNA. These data suggest that while IL-1R1 was increased in 6-OHDA-treated animals and reduced following shRNA injection, the neurodegeneration induced by 6-OHDA was not mediated through IL-1R1.

Changes in matrix metalloprotease activity and progranulin levels may contribute to the pathophysiological function of mutant leucine‐rich repeat kinase 2

Increasing evidence suggests that Parkinson's disease (PD)-linked Leucine-rich repeat kinase 2 (LRRK2) has a role in peripheral and brain-resident immune cells. Furthermore, dysregulation of the anti-inflammatory, neurotrophic protein progranulin (PGRN) has been demonstrated in several chronic neurodegenerative diseases. Here we show that PGRN levels are significantly reduced in conditioned medium of LRRK2(R1441G) mutant mouse fibroblasts, leukocytes, and microglia, whereas levels of proinflammatory factors, like interleukin-1β and keratinocyte-derived chemokine, were significantly increased. Decreased PGRN levels were also detected in supernatants of cultured human fibroblasts isolated from presymptomatic LRRK2(G2019S) mutation carriers, while mitochondrial function was unaffected. Furthermore, medium levels of matrix metalloprotease (MMP) 2 increased, whereas MMP 9 decreased in LRRK2(R1441G) mutant microglia. Increased proteolytic cleavage of the MMP substrates ICAM-5 and α-synuclein in synaptoneurosomes from LRRK2(R1441G) mutant mouse brain indicates increased net synaptic MMP activity. PGRN levels were decreased in the cerebrospinal fluid of presymptomatic LRRK2 mutant mice, whereas PGRN levels were increased in aged symptomatic mutant mice. Notably, PGRN levels were also increased in the cerebrospinal fluid of PD patients carrying LRRK2 mutations, but not in idiopathic PD patients and in healthy control donors. Our data suggest that proinflammatory activity of peripheral and brain-resident immune cells may particularly contribute to the early stages of Parkinson's disease caused by LRRK2 mutations.

Parkinson's Disease Biomarkers: Resources for Discovery and Validation

Parkinson's disease (PD) is currently diagnosed using clinical features. While experienced neurologists can typically diagnose PD with 70–90% accuracy, there are many situations early in the course of the disease when clinical diagnosis is less precise. There is a paucity of objective measures that could be employed to improve the diagnosis, stratify patients by subtypes, and track underlying disease progression. Development of innovative new therapies that slow or stop the progression of the disease would be accelerated by such objective biomarkers. Various imaging modalities including specific dopaminergic markers and morphometric measures are available, but have not been firmly established as markers for these purposes. Markers that can be detected in easily obtainable biofluids would be ideal for these purposes and would increase the power to detect the effect of therapeutic agents in a shorter time with reduced cost. Unbiased exploratory examination of panels of RNAs, both mRNA and noncoding RNAs, exosomes, proteins, antibodies, and metabolites in small individual cohorts have yielded promising candidate markers that yearn for replication in independent cohorts (Kroksveen et al., 2011). More targeted approaches to identify biomarkers based on PD pathophysiology have shown disease-related proteins as strong candidates. Alpha-synuclein is decreased in the cerebrospinal fluid of PD patients (Hong et al., 2010), although significant overlap with controls makes it less useful in assisting individual diagnosis. Other potential protein candidates include DJ-1 and inflammatory cytokines. Post-translationally modified forms of these proteins may improve the biomarker specificity. Most of these studies have utilized CSF (Parnetti et al., 2013), although increased oxidized DJ-1 in PD patients was detected in blood (Saito et al., 2009) and epidermal growth factor levels in plasma has been shown to predict cognitive decline in PD (Chen-Plotkin et al., 2011). Ultimately one could predict that a panel of multiple biochemical markers could be combined to increase the accuracy of diagnosis and disease progression. Heterogeneity of patient populations and lack of standardization of collection methods may contribute to the inconsistent and variable observations in the literature. A major need in the field is the availability of biospecimens from well-characterized cohorts for discovery of new biomarkers and validation. In response to these needs, three major programs have been launched. The Parkinson's Progression Marker Initiative (PPMI) is a prospective study of 400 newly diagnosed PD patients and 200 controls that will be followed over 5 years and collect extensive clinical (motor and non-motor information), imaging, and biosample (blood, cerebrospinal fluid, DNA/RNA from blood, urine) information from all 600 subjects at sites around the world (Marek, 2011). PPMI expanded to include a prodromal cohort of 100 individuals at risk for developing PD to develop biomarkers that are present prior to the onset of clinical motor symptoms. The Fox Investigation for New Discovery of Biomarkers (BioFIND) is a study supported in collaboration with National Institute of Neurological Diseases and Stroke (NINDS) focused on novel biomarker discovery by enrolling 120 rigorously defined clinically typical PD in mid-stage and 120 age- and gender-matched controls at one timepoint in US sites. BioFIND was launched to serve as a platform to test new biomarkers in somewhat narrow spectrum of clinically typical PD in moderate stages to maximize the chance of discovering differences in a less heterogeneous population. The Parkinson's Disease Biomarker Program (PDBP) is a program designed to support new and existing biomarker cohorts that collect biospecimens using standardized protocols. Biospecimens and data from all above studies are available to the research community. Through standardization and coordination of data and sample collection, we are optimistic that new markers will emerge which will assist in clinical trials and ultimately result in improved management of the disease.

English

Introduction Halogenative and nitrosative stress are two types of oxidative stress that have been proposed as pathogenic mechanisms in Parkinson’s disease (PD). They can be caused by overstimulation of phagocytes. This hypothesis discusses serum protein halogenation and nitrosylation as traits of maintained overstimulation of blood phagocytes in sporadic Parkinson’s disease. Hypothesis I hypothesize that maintained phagocyte overstimulation leads to both halogenative and nitrosative stress in PD, which are present in the serum and cerebrospinal fluid of patients. These types of oxidative stress could modify proteins related to the pathogenesis of PD. Evaluation of hypothesis It has been detected that the presence of halogenative stress in the serum and, to a lesser extent, cerebrospinal fluid of Parkinsonian patients leads to excess of advanced oxidized protein products. On the other hand, nitrosative stress is also present in serum and cerebrospinal fluid of patients with early PD, characterized by the selective increase of 3-nitrotyrosine proteins other than nitroalbumin and free 3-nitrotyrosine. Nitrosylation stress accompanies modification of the sites of nitrosylation of αsynuclein in these patients, characterized by dominant nitrosylation of tyrosine 125/136 residues. Conclusion Since metabolism of advanced oxidized protein products and 3nitrotyrosine proteins has been associated with phagocytic overstimulation, this pathological alteration could play a pathogenic role in sporadic PD. Our observations also lead to the hypothesis that serum level of advanced oxidized protein products is a prognostic marker for PD duration, and these oxidized proteins could participate in neuroinflammation. Besides, the evaluation of nitrosative stress through enhanced levels of 3-nitrotyrosine proteins in serum and cerebrospinal fluid without changes in nitroalbumin, together with the profile of tyrosine nitrosylation of serum α-synuclein characterized by dominant nitrosylation of Tyr125/136, could serve for the diagnosis of sporadic PD. Nitroα-synuclein is a main component of Lewy bodies, hallmarks of the disease, and serum nitro-α-synuclein could represent a pathogenic factor in PD. Introduction Oxidative stress is defined as an imbalance between the production of reactive oxygen and nitrogen species (ROS/RNS) and antioxidant mechanisms, and it is considered as an important pathogenic mechanism in Parkinson’s disease (PD)1–4. Halogenation is a type of oxidative stress induced by phagocytic overstimulation, leading to excess of several molecules such as advanced oxidized protein products (AOPPs). These products originate as a result of the action of free radicals such as chloramines and hypochlorous acid (HOCl) on proteins, and they are augmented in several diseases5. This halogenative process could also modify free amines such as tyrosine, leading to the formation of free 3chlorotyrosine, as shown in Figure 1. Another type of oxidative stress is nitrosylation, where oxidative modifications of proteins are secondary to excess of nitric oxide. Nitrosylation of tyrosine leading to 3-nitrotyrosine proteins or free 3-nitrotyrosine is the most prominent change, and this type of oxidative stress has been associated with human pathologies6, and it is recognized as a salient feature of diverse α-synucleinopathies such as PD and Lewy body dementia7. In PD, protein tyrosine nitrosylation has been demonstrated to represent a pathogenic mechanism, and nitrosylated tyrosine residues of diverse proteins such as neurofilaments or α-synuclein are detected in the brain proteinaceous aggregates or Lewy bodies, hallmarks of the disease8. Moreover, nitrating agents generated by dopamine are known to promote α-synuclein aggregation9, a critical step for the formation of Lewy bodies. Biochemically, nitrosylated proteins and amines can be formed through different pathways (Figure 1). The formation of peroxynitrite (ONOO–) through nitric oxide and superoxide anion is the main pathway10. However, other nitrating pathways related to phagocyte overstimulation exist. For instance, myeloperoxidase (MPO) activity and nitric oxide excess from macrophages or brain microglia yield nitrosylated proteins and nitrotyrosines11. This hypothesis discusses if protein halogenation * Corresponding author Email: efespejo@us.es University of Seville, Department of Medical Physiology, Laboratory of Molecular Neurology and Neurophysiology, E-41009 Seville, Spain

S.03.02 Neuronal stem cells in Parkinson's disease

Parkinson's Disease (PD) and Multiple System Atrophy (MSA) are neurodegenerative diseases characterized neuropathologically by alpha-synuclein accumulation in brain cells. This accumulation is hypothesized to contribute to constitutive neuroinflammation, and to participate in the neurodegeneration. Cytokines, which are the main inflammatory signalling molecules, have been identified in blood and cerebrospinal fluid of PD patients, but studies investigating the human brain levels are scarce. It is documented that neurotrophins, necessary for survival of brain cells and known to interact with cytokines, are altered in the basal ganglia of PD patients. In regards to MSA, no major study has investigated brain cytokine or neurotrophin protein expression.Here, we measured protein levels of 18 cytokines (IL-2, 4–8, 10, 12, 13, 17, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1α and 1β, TNF-α) and 5 neurotrophins (BDNF, GDNF, bFGF, PDGF-BB, VEGF) in the dorsomedial prefrontal cortex in brains of MSA and PD patients and control subjects. We found altered expression of IL-2, IL-13, and G-CSF, but no differences in neurotrophin levels. Further, in MSA patients we identified increased mRNA levels of GSK3β that is involved in neuroinflammatory pathways. Lastly, we identified increased expression of the neurodegenerative marker S100B, but not CRP, in PD and MSA patients, indicating local rather than systemic inflammation. Supporting this, in both diseases we observed increased MHC class II+ and CD45+ positive cells, and low numbers of infiltrating CD3+ cells. In conclusion, we identified neuroinflammatory responses in PD and MSA which seems more widespread in the brain than neurotrophic changes.

‘‘70th Birthday Professor Riederer’’ Induction of glial cell line-derived and brain-derived neurotrophic factors by rasagiline and (−)deprenyl: a way to a disease-modifying therapy?

Neuroprotection has been proposed in neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, to delay or halt disease progression or reverse neuronal deterioration. The inhibitors of type B monoamine oxidase (MAO), rasagiline and (-)deprenyl, prevent neuronal loss in cellular and animal models of neurodegenerative disorders by intervening in the death signal pathway in mitochondria. In addition, rasagiline and (-)deprenyl increase the expression of anti-apoptotic Bcl-2 protein family and neurotrophic factors. Neurotrophic factors, especially glial cell line-derived neurotrophic factor (GDNF) and brain-derived derived neurotrophic factor (BDNF), are required not only for growth and maintenance of developing neurons, but also for function and plasticity of distinct population of adult neurons. GDNF and BDNF have been reported to reduce Parkinson and Alzheimer's diseases, respectively. GDNF protects the nigra-striatal dopamine neurons in animal models of Parkinson's disease, and its administration has been tried as a disease-modifying therapy for parkinsonian patients. However, the results of clinical trials have not been fully conclusive and more practical ways to enhance GDNF levels in the targeted neurons are essentially required for future clinical application. Rasagiline and (-)deprenyl induced preferentially GDNF and BDNF in cellular and non-human primate experiments, and (-)deprenyl increased BDNF level in the cerebrospinal fluid of parkinsonian patients. In this paper, we review the induction of GDNF and BDNF by these MAO inhibitors as a strategy of neuroprotective therapy. The induction of prosurvival genes is discussed in relation to a possible disease-modifying therapy with MAO inhibitors in neurodegenerative disorders.